Autoimmune diseases afflict 7-10% of Americans, and their incidence is rising. APECED patients have a multi-organ autoimmune disorder, the study of which has yielded important insights into immunological tolerance and autoimmune diseases more generally. The mutated gene underlying APECED encodes Aire, a large protein with several structural domains characteristic of a transcriptional regulator. Mutation of the locus encoding murine Aire also resulted in multi-organ autoimmunity. Studies on this model revealed Aire to operate primarily within a rare population of thymic stromal cells by inducing the expression of a large repertoire of peripheral-tissue antigens, or PTAs. Consequently, as differentiating T cells percolate through the thymus, those capable of responding to such PTAs avoid clonal deletion; when they emerge into the periphery and encounter cognate antigen, autoimmunity ensues. Aire's molecular mechanisms remain enigmatic. It controls gene expression but does not appear to act as a conventional transcription factor. Rather, it seems to be highly cooperative in its activities, participating in large multi-protein complexes that incorporate proteins of diverse function, involved in nuclear transport, chromatin binding/structure, transcriptional regulation or pre-mRNA processing. Our preliminary studies highlighted an unexpected Aire partner: DNA-dependent protein kinase (DNA-PK), usually associated with the repair of DNA double-stranded breaks (DSBs) via non-homologous end joining, but also recently implicated in the control of transcriptional elongation. The goal of this proposed project is to elucidate how Aire and DNA-PK interact to promote transcription of a large, but select, portion of the genome specifically in rare thymic stromal cells. Proposed studies aim to: 1) Structurally and functionally define the [Aire/DNA-PK]-containing complex(es), combining sequential affinity-purifications, candidate partner co-precipitations, shRNA knockdowns in cells, and gene-knockout mice. 2) Determine whether Aire promotes the generation/stability of DNA DSBs, pursuing the hypothesis (issuing from our preliminary studies) that it operates like the anti-cancer drug etoposide to inhibit DNA topoisomerase-2 from resolving DNA cuts after introducing them to relieve the torsional stress associated with transcription. 3) Determine whether Aire impacts on transcription via the histone eviction machinery, testing the hypothesis, that it promotes the recruitment and/or effectiveness of a complex (including DNA-PK, PARP1, TOP2, FACT, H2AX) responsible for disassembling and re-assembling nucleosomes as the transcriptional machinery progresses along DNA. These studies should yield new insights into the molecular mechanisms by which Aire controls immunological tolerance. Besides APECED, this mode of T cell tolerization is thought to play an important role in common autoimmune diseases, notably type-1 diabetes and myasthenia gravis. Successful intervention in the autoimmune disease of Aire-deficient mice with the cancer drug, etoposide, or allied drugs would represent proof-of-principle that this tolerance pathway can be targeted for therapeutic benefit.